Defective plasma membrane assembly in yeast secretory mutants.

Yeast mutants that are conditionally blocked at distinctive steps in secretion and export of cell surface proteins have been used to monitor assembly of integral plasma membrane proteins. Mutants blocked in transport from the endoplasmic reticulum (sec18), from the Golgi body (sec7 and sec14), and in transport of secretory vesicles (sec1) show dramatically reduced assembly of galactose and arginine permease activities. Simultaneous induction of galactose permease and alpha-galactosidase (a secreted glycoprotein) in sec mutant cells at the nonpermissive temperature (37 degrees C) shows that both activities accumulate and can be exported coordinately when cells are returned to the permissive temperature (24 degrees C) in the presence or absence of cycloheximide. Plasma membrane fractions isolated from sec mutant cells radiolabeled at 37 degrees C have been analyzed by two-dimensional sodium dodecyl sulfate-gel electrophoresis. Although most of the major protein species seen in plasma membranes from wild-type cells are not efficiently localized in sec18 or sec7, several of these proteins appear in plasma membranes from sec1 cells. These results may be explained by contamination of plasma membrane fractions with precursor vesicles that accumulate in sec1 cells. Alternatively, some proteins may branch off during transport along the secretory pathway and be inserted into the plasma membrane by a different mechanism.     

Melibiose transport system in Lactobacillus plantarum.

Lactobacillus plantarum ATCC 8014 grew on melibiose at 30 C, but not at 37 C, although it grew on galactose or lactose at either temperature. ATCC 8014 grown on lactose at 30 or 37 C accumulated melibiose slowly, suggesting that melibiose may partly be transported by a lactose transport system. A lactose-negative mutant, NTG 21, derived from ATCC 8014 was isolated. The mutant was totally deficient in lactose transport, but retained normal melibiose transport activity. In NTG 21, the melibiose transport activity was induced by melibiose at 30 C, but not at 37 C. The transport activity itself was found to be stable for at least 3 hr at 37 C, suggesting that the induction process in the cytoplasm rather than the inducer entrance is temperature-sensitive in the organism. The organism also failed to form alpha-galactosidase at 37 C when grown on melibiose. The enzyme synthesis, however, was induced by galactose in NTG 21 (and also by lactose in ATCC 8014) even at 37 C, indicating that the induction of the enzyme is essentially not temperature-sensitive. In NTG 21, melibiose transport system and alpha-galactosidase were induced by galactose, melibiose and o-nitrophenyl-alpha-D-galactopyranoside when the strain was grown at 30 C. Raffinose induced melibiose transport system only a little, while it was a good inducer for alpha-galactosidase. Inhibition studies revealed that galactose may be a weak substrate of the melibiose transport system; no inhibition was demonstrated with lactose and raffinose.     

Conditions of formation, purification, and characterization of an alpha-galactosidase of Trichoderma reesei RUT C-30.

Trichoderma reesei RUT C-30 formed an extracellular alpha-galactosidase when it was grown in a batch culture containing lactose or locust bean gum as a carbon source. Short-chain alpha-galactosides (melibiose, raffinose, stachyose), as well as the monosaccharides galactose, dulcitol, arabinose, and arabitol, also induced alpha-galactosidase activity both when they were used as carbon sources (at a concentration of 1%) in batch cultures and in resting mycelia (at concentrations in the millimolar range). The addition of 50 mM glucose did not affect the induction of alpha-galactosidase formation by galactose. alpha-Galactosidase from T. reesei RUT C-30 was purified to homogeneity from culture fluids of galactose-induced mycelia. The active enzyme was a 50 +/- 3-kDa, nonglycosylated monomer which had an isoelectric point of 5.2. It was active against several alpha-galactosides (p-nitrophenyl-alpha-D-galactoside, melibiose, raffinose, and stachyose) and galactomannan (locust bean gum) and was inhibited by the product galactose. It released galactose from locust bean gum and exhibited synergism with T. reesei beta-mannanase. Its activity was optimal at pH 4, and it displayed broad pH stability (pH 4 to 8). Its temperature stability was moderate (60 min at 50 degrees C resulted in recovery of 70% of activity), and its highest level of activity occurred at 60 degrees C. Its action on galactomannan was increased by the presence of beta-mannanase.     

Transcription of the phosphoglycerate kinase gene of Saccharomyces cerevisiae increases when fermentative cultures are stressed by heat-shock

The single gene for phosphoglycerate kinase (PGK) in the haploid genome of Saccharomyces cerevisiae is expressed to a very high level in cultures fermenting glucose. Despite this it responds to heat-shock. When S. cerevisiae growing exponentially on glucose media was shifted from 25 degrees C to 38 degrees C transient increases of 6-7-fold in cellular PGK mRNA were observed. This elevation in PGK mRNA still occurred in the presence of the protein-synthesis inhibitor cycloheximide, but was not observed in cells bearing the rna1.1 mutation. From the kinetics of continuous labelling of PGK mRNA, relative to the labelling of other RNAs in the same cultures whose levels do not alter with heat-shock, it was shown that the elevation in PGK mRNA in response to temperature upshift reflects primarily an increased synthesis of this mRNA and not an alteration of its half-life. PGK mRNA synthesis is therefore one target of a response mechanism to thermal stress. Synthesis of PGK enzyme in glucose-grown cultures is efficient after mild (25 degrees C to 38 degrees C) or severe (25 degrees C to 42 degrees C) heat-shocks. Following the severe shock, the synthesis of most proteins is abruptly terminated, but synthesis of PGK and a few other glycolytic enzymes continues at levels comparable to the levels of synthesis of most of those proteins dramatically induced by heat (heat-shock proteins). Cells that overproduce PGK due to the presence of multiple copies of the PGK gene on a high-copy-number plasmid continue their overproduction of this enzyme during severe thermal stress. Therefore PGK mRNA is both elevated in level in response to heat-shock and translated efficiently at supra-optimal temperatures.     

Regulation of beta-D-galactosidase synthesis in Candida pseudotropicalis.

Regulation of lactose (beta-D-galactosidase) synthesis in the lactose-utilizing yeast Candida pseudotropicalis was studied. The enzyme was inducible by lactose and galactose. When grown on these sugars the enzyme level of the yeast was 20 times or higher than when grown on glycerol. The Km and optimal pH were similar for the lactase induced either by lactose or galactose. The hydrolysis of o-nitrophenyl-beta-D-galactopyranoside by the lactase was inhibited by galactose and several analogs and galactosides, but not by glucose. Lactose uptake activity observed in lactose-grown cells was very reduced in cells grown on glucose or galactose. Glucose repressed the induction of lactase, but not the metabolic system for galactose utilization. In continuous culture on lactose medium at dilution rates below 0.2 h-1 the specific lactase activity was higher than in batch cultures and decreased with increases in dilution rate. Lactase was induced by pulses of lactose and galactose in cells growing on glucose, but only at low dilution rates were the steady-state concentration of glucose was very low.     

Abortive infection of the virulent phage 9NA in a fatty acid auxotroph of Salmonella typhimurium: effect of fatty acid supplementation

A conditional (temperature sensitive) fatty acid biosynthetic mutant (fabB2) of Salmonella typhimurium does not support the development of the virulent bacteriophage 9NA even at permissive temperature (30 degrees C). A limited amount of phage DNA synthesis takes place at this temperature. When the fatty acid composition of the host membrane is altered by growing the cells at 37 degrees C in the presence of exogenous unsaturated fatty acid, differential expression of phage genes was observed. Phage specific lysozyme is induced when the cultures are supplemented with elaidic, palmitelaidic, linoleic and linolelaidic acids but not with oleic and plamitoleic acids. However, in no case were infective particles produced. Under conditions where no lysozyme is synthesized the infected cells increase in length and become filamentous.     

Overexpression of the actin gene is associated with the morphogenesis of Candida albicans

A progressive increase in the synthesis of actin mRNA was observed by Northern blot analysis, when cells were induced to form germ tubes at 37 degrees C by N-acetyl-D-glucosamine. Presence of trifluoperazine, a calmodulin inhibitor, or incubation of cells at 25 degrees C, or by replacing N-acetyl-D-glucosamine with glucose which inhibited germ tube formation lowered this synthesis. Furthermore, in vitro translation of total RNA revealed an increase in the synthesis of actin (45 kDa) during germ tube formation. These results suggest for the first time that the expression of actin gene is regulated during morphogenesis of C. albicans.     

Insulin processing and signal transduction in rat adipocytes

A glycine-HCl buffer (glycine, 50 mM/NaCl, 0.15 M/HCl, pH 3.5) was used to strip insulin bound to adipocyte cell surfaces. Adipocytes retained their integrity in the glycine buffer and their binding capacity for [125I]iodoinsulin could be completely recovered on transfer of the cells to physiological media. At 37 degrees C, [125I]iodoinsulin binds rapidly to plasma membrane receptors; maximal binding occurs within 10 min. At this temperature, the initial binding is followed by rapid internalization, degradation of the hormone and subsequent loss of label. Insulin treatment, at 37 degrees C, induced internalization of 37% of the plasma membrane insulin receptors. Phenylarsine oxide (PAO), a confirmed inhibitor of protein internalization, allowed insulin binding but completely inhibited degradation of the hormone. Monensin, a carboxylic ionophore which impairs uncoupling hormone-receptor complexes, effectively restricted insulin degradation over short time periods (less than 30 min). Addition of monensin to insulin-stimulated cells did not impair D-glucose uptake. It has previously been reported that PAO inhibits hexose transport through the direct interaction with the glucose transporters and low concentrations of PAO (1 microM) transiently inhibit insulin-stimulated glucose uptake. This recovery phenomenon was again observed when PAO was added to insulin-stimulated, monensin-treated adipocytes. The data suggests that lysosomal degradation of insulin is not requisite for signal transduction.     

RNA Synthesis in Yersinia pestis During Growth Restriction in Calcium-Deficient Medium

Yersinia pestis requires 2.5 mM Ca(2+) for growth at 37 degrees C but not at 26 degrees C. After a shift from 26 to 37 degrees C in a Ca(2+)-deficient medium, an ordered series of metabolic alterations occur which result in transition from a growing cell to a viable but non-proliferating cell. The earliest known alteration in normal metabolism associated with this transition is a termination of net RNA synthesis. Competitive RNA/DNA hybridizations with uniformly labeled RNA and stable RNA competitor indicated identical mRNA to stable RNA ratios in growing cells and non-proliferating Ca(2+)-deprived cells. Similar hybridizations with pulse-labeled RNA demonstrated that growing cells synthesized 57% mRNA, 37% rRNA, and 5% tRNA, whereas Ca(2+)-deprived cells synthesized 95% mRNA, 4.7% rRNA, and 0.7% tRNA. After addition of radioactive uracil and rifampin to growing and Ca(2+)-deprived cells, decay of approximately 40 and 90% of the newly synthesized RNA was found for growing and Ca(2+)-deprived cells, respectively. The half-life of the mRNA was found to be 1.5 min for growing cells and 4.5 min for Ca(2+)-deprived cells. Y. pestis elicited increases in the levels of guanosine tetraphosphate and guanosine pentaphosphate in response to amino acid deprivation and yielded transient increases in the levels of these phosphorylated nucleotides after a shift from 26 to 37 degrees C. These increases were independent of Ca(2+) availability and preceded the alteration in RNA synthesis by more than 1 h. The levels of these phosphorylated nucleotides then stabilized at about 80 and 40 pmol for Ca(2+)-deprived and Ca(2+)-supplemented cultures, respectively, and did not increase further in the Ca(2+)-deprived culture at the time corresponding to the reduction in stable RNA synthesis. These findings indicate that the early lesion in RNA synthesis associated with the growth restriction of Ca(2+)-deprived Y. pestis reflects a block in stable RNA synthesis and that this effect is not mediated by guanosine tetraphosphate or guanosine pentaphosphate.     

Cultured human skin fibroblasts modify their plasma membrane lipid composition and fluidity according to growth temperature suggesting homeoviscous adaptation at hypothermic (30 degrees C) but not at hyperthermic (40 degrees C) temperatures.

Mammalian cell metabolism is responding to changes in temperature. Body temperature is regulated around 37 degrees C, but temperatures of exposed skin areas may vary between 20 degrees C and 40 degrees C for extended periods of time without apparent disturbance of adequate cellular functions. Cellular membrane functions are depending from temperatures but also from their lipid environment, which is a major component of membrane fluidity. Temperature-induced changes of membrane fluidity may be counterbalanced by adaptive modification of membrane lipids. Temperature-dependent changes of whole cell- and of purified membrane lipids and possible homeoviscous adaptation of membrane fluidity have been studied in human skin fibroblasts cultured at 30 degrees C, 37 degrees C, and 40 degrees C for ten days. Membrane anisotropy was measured by polarized fluorescence spectroscopy using TMA-DPH for superficial and DPH for deeper membrane layers. Human fibroblasts were able to adapt themselves to hypothermic temperatures (30 degrees C) by modifying the fluidity of the deeper apolar regions of the plasma membranes as reported by changes of fluorescence anisotropy due to appropriate changes of their plasma membrane lipid composition. This could not be shown for the whole cells. At 40 degrees C growth temperature, adaptive changes of the membrane lipid composition, except for some changes in fatty acid compositions, were not seen. Independent from the changes of the membrane lipid composition, the fluorescence anisotropy of the more superficial membrane layers (TMA-DPH) increased in cells growing at 30 degrees C and decreased in cells growing at 40 degrees C.     

Induction of Galactokinase in Saccharomyces cerevisiae: Kinetics of Induction and Glucose Effects

The induced synthesis of galactokinase and the repressing effects of glucose on this synthesis have been investigated in whole yeast cells rendered permeable by treatment with dimethyl sulfoxide. It was found that the induction response of uninduced cells to galactose is clearly dependent on the nature of the carbon source upon which the culture was grown prior to exposure to galactose. Glucose-grown cells exhibited a long lag before induction, whereas lactate-grown cells exhibited induced synthesis within 8 min. A concentration of 0.5% galactose was found to be optimal for induction. The addition of glucose to yeast cultures growing on galactose resulted in a severe transient repression of synthesis which was followed by a resumed rate of synthesis characteristic of a weaker permanent catabolite repression. Neither 2-deoxygalactose nor fucose acted as gratuitous inducers of the pathway, nor did they serve as a substrates for galactokinase.     

Survival of biological cells deformed in a narrow gap

Recent studies show that during slow freezing of biological cells, the cells may be also injured by not only chemical damage but also mechanical damage induced by ice crystal compression. A new experimental procedure is developed to quantify cell destruction by deformation with two parallel surfaces. The viability of cells (prostatic carcinoma cells, 17.5 microns in mean diameter) is measured as a function of gap size ranging from 3.5 microns to 16.2 microns at 0 degree C, 23 degrees C and 37 degrees C. The viability at a smaller gap size is significantly lower at 37 degrees C than at 23 degrees C, while the difference between 0 degree C and 23 degrees C is much smaller. This suggests that deformation damage is related to the deformation of the cytoskeleton rather than the mechanical properties of the lipid membrane.     

Effects of temperature on basal and insulin-stimulated glucose transport activities in fat cells. Further support for the translocation hypothesis of insulin action

Effects of temperature on glucose transport in fat cells were studied. In this system, the basal (no insulin) glucose transport activity was higher at approximately 25-30 degrees C than at 37 degrees C, as previously reported (Vega, F. V., and Kono, T. (1979) Arch. Biochem. Biophys. 192, 120-127). The stimulatory effect of low temperature (or the insulin-like effect) was reversible and apparently required metabolic energy for both its forward and reverse reactions. By lowering the ATP level with 2,4-dinitrophenol, one could separately determine the insulin-like stimulatory effect of low temperature and its inhibitory effect on the transport process itself. The maximum level of stimulation by low temperature was greater at 10 degrees C than at 25-30 degrees C, but the rate of stimulation was considerably slower at 10 degrees C than at 25-30 degrees C. When cells were exposed to low temperature, the glucose transport activity in the plasma membrane-rich fraction was increased, while that in the Golgi-rich fraction was decreased. The Arrhenius plot of the basal glucose transport activity determined in the presence of dinitrophenol was apparently linear from 10 to 37 degrees C and parallel to that of the plus insulin activity measured either in the presence or absence of dinitrophenyl. Insulin itself slowly stimulated the glucose transport activity at 10 degrees C. These results are consistent with the view that (a) low temperature, like insulin, induces translocation of the glucose transport activity from an intracellular storage site to the plasma membrane, (b) insulin stimulates glucose transport activity without changing its activation energy, and (c) subcellular membranes do not entirely stop their movement at a low temperature, e.g, at 10 degrees C.     

Regulation of mechanical stability of erythrocytes by serum immunoglobulins

Autologic plasma protects human erythrocytes from hemolysis induced by their suction through glass microfiber filters. The protective effect is related to the protein fraction with molecular mass above 100 kD and reproduced by gamma-globulin. The action of proteins is abolished after heating the erythrocytes above 45 degrees C as well as in the presence of galactose and ribose but not glucose, mannose and lactose. It is suggested that an increase in mechanical stability of erythrocytes is caused by interaction of immunoglobulins with glycoproteid and glycolipid membrane receptors and mediated by the changes of structural state of the cytoskeleton.     

Plasma membrane expansion terminates in Saccharomyces cerevisiae secretion-defective mutants while phospholipid synthesis continues.

Phospholipid synthesis activity and plasma membrane growth have been studied in the Saccharomyces cerevisiae temperature-sensitive, secretion-defective mutants isolated by Novick and Schekman (Proc. Natl. Acad. Sci. U.S.A. 76:1858-1862, 1979; Novick et al., Cell 21:205-215, 1980). The mutants, sec1 through sec23, do not grow at 37 degrees C and exhibit lower rates of phospholipid synthesis than does the wild-type strain X2180. None of the mutants exhibits a decline in lipid synthesis rapid enough to explain secretion failure. Plasma membrane growth was assessed indirectly by examining the osmotic sensitivity of spheroplasts derived from cultures transferred from 24 to 37 degrees C. Spheroplasts from the normal-growing strain X2180 exhibited a small rapid increase in osmotic sensitivity and stabilized at a more sensitive state. Spheroplasts from the sec mutants exposed to the same temperature shift exhibited progressively increasing osmotic sensitivity. Cycloheximide treatment prevented progressive increases in osmotic fragility. These data are compatible with the hypothesis that plasma membrane expansion is restricted in the sec mutants. During incubation at 37 degrees C, the accumulation of intracellular materials within the no-longer expanding plasma membrane exerts osmotic stress on the membrane, increasing with time. The gene products defective in Novick and Schekman's sec mutants appear to be required for both extracellular protein secretion and plasma membrane growth in yeast cells.     

Transport of a fluorescent phosphatidylcholine analog from the plasma membrane to the Golgi apparatus

We have examined the internalization and degradation of a fluorescent analog of phosphatidylcholine after its insertion into the plasma membrane of cultured Chinese hamster fibroblasts. 1-acyl-2-(N-4- nitrobenzo-2-oxa-1,3-diazole)-aminocaproyl phosphatidylcholine (C6-NBD- PC) was incorporated into the cell surface by liposome-cell lipid transfer at 2 degrees C. The fluorescent lipid remained localized at the plasma membrane as long as the cells were kept at 2 degrees C; however, when the cells were warmed to 37 degrees C, internalization of some of the fluorescent lipid occurred. Most of the internalized C6-NBD- PC accumulated in the Golgi apparatus although a small amount was found randomly distributed throughout the cytoplasm in punctate fluorescent structures. Internalization of the fluorescent lipid at 37 degrees C was blocked by the presence of inhibitors of endocytosis. Incubation of cells containing C6-NBD-PC at 37 degrees C resulted in a rapid degradation of the fluorescent lipid. This degradation occurred predominantly at the plasma membrane. The degradation of C6-NBD-PC resulted in the release of NBD-fatty acid into the medium. We have compared the internalization of the fluorescent lipid with that of a fluorescent protein bound to the cell surface. Both fluorescent lipid and protein remained at the plasma membrane at 2 degrees C and neither were internalized at 37 degrees C in the presence of inhibitors of endocytosis. However, when incubated at 37 degrees C under conditions that permit endocytosis, the two fluorescent species appeared at different intracellular sites. Our data suggest that there is no transmembrane movement of C6-NBD-PC and that the fluorescent probe reflects the internalization of the outer leaflet of the plasma membrane lipid bilayer. The results are consistent with the Golgi apparatus as being the primary delivery site of phospholipid by bulk membrane movement from the plasma membrane.     

Induction of D-galactonate dehydratase in Mycobacterium butyricum

The induced synthesis of D-galactonate dehydratase in Mycobacterium butyricum has been studied initially after addition or removal of inductor or inhibitor. The enzyme was induced by galactonate and galactose; the system reached half-maximal effect of synthesis at 3.3 mM of galactonate. The lag of about 30 min between the addition of the inductor and the appearance of the enzyme at 37 degrees C was noted. The lag was dependent on temperature and independent of inductor concentration. After the withdrawal of the inductor the expression of a supposed galactonate dehydratase-coding messenger takes place which can be blocked by streptomycin or chloramphenicol. Both the messenger (the mean life of about 38 min) and the enzyme appeared relatively stable. The enzyme synthesis was found to be under strong catabolite repression caused by glucose and several other compounds and cyclic AMP failed to increase the enzyme synthesis or to overcome the repression. Zinc ions at concentration below 1 mM proved to have no effect on the enzyme synthesis but inhibited the enzyme itself that can be restored by EDTA.